The cultured vascular smooth muscle cell system provides an excellent model for studying many of the physiologic and biologic properties of vascular smooth muscle. The purpose of this investigation is to utilize this system to study the contractile apparatus in vascular smooth muscle applying recombinant DNA techniques that have proven of great value in studying skeletal and cardiac muscle contractile systems. During the initial part of this project, Dr. Taubman will learn the recombinant DNA techniques under the sponsorship of Dr. Bernardo Nadal-Ginard, a leader in the use of DNA cloning for the study of the skeleted and cardiac muscle contractile systems. A library of cDNA clones will be derived from rat aortic smooth muscle cell mRNA and clones will be selected containing inserts coding for myosin heavy and light chains, Alpha-actin, and tropomyosin. These inserts will be isolated and restriction maps generated from which nucleotide sequences will be obtained. These will provide detailed information concerning the structure of the smooth muscle contractile proteins and allow for comparisons with similar genes derived from skeletal and cardiac muscle. The cDNA inserts will be used to investigate the existence of multigene families encoding the smooth muscle contractile proteins, analagous to those found in skeletel muscle. During the latter part of the project, Dr. Taubman will employ the isolated cDNA inserts as hybridization probes to study contractile protein mRNA expression in cultured vascular smooth muscle cells. This work will be performed under the joint sponsorship of Dr. Nadal-Ginard and Dr. R. Wayne Alexander. Particular attention will be given to identifying the specific contractile protein genes expressed during phenotypic modulation of cultured vascular smooth muscle cells from contractile to non-contractile states. Changes in mRNA expression will be analyzed during prolonged exposure of vascular smooth muscle cells to vasoactive hormones such as angiotensin II. mRNA expression will also be compared in cells derived from spontaneously hypertensive rats and from cells derived from different types of vascular and non-vascular smooth muscle. These studies should provide important insights into the mechanisms governing vascular reactivity at the cellular level.